Automated method and system for the analysis of total dietary fiber

ABSTRACT

The invention consists of a method for determining Total Dietary Fiber (TDF) and its sub-fractions, Insoluble Dietary Fiber (IDF) and Soluble Dietary Fiber (SDF) in food and feed samples which utilizes flexible reaction/filtration containers that can be divided into one or more sections for capturing the IDF and SDF fractions separately or for capturing TDF in its entirety. Each container is fashioned as a bag that can be temporarily sealed in multiple locations to create multiple sections and is made of non-porous and porous material. Use of these containers eliminates the need for problematic transfers of mixtures from beaker to filter, and vastly improves the filtration process.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 13/392,413, entitled“An Automated Method And System For The Analysis Of Total Dietary Fiber”filed Feb. 24, 2012, which claims priority from U.S. Provisional PatentApplication Ser. No. 61/236,729, filed Aug. 25, 2009. The entirety ofall applications is incorporated hereby reference.

FIELD OF THE INVENTION

This invention relates generally to the analysis of fiber and morespecifically to analyzing dietary fiber with the use of a flexiblereaction/filtration container that can be divided into one or moresections.

BACKGROUND OF THE INVENTION

This invention is directed to an automated Total Dietary Fiber (TDF)analysis system which exhibits improved efficiency in filtration, laborand time, and eliminates the glassware and the associated glasswarecleanup.

A variety of methods have been developed for the analysis of fiber infeeds and foods. Generally accepted methods for analyzing feeds inanimal nutrition are Crude Fiber (AOAC Method 962.09) and NeutralDetergent Fiber and Acid Detergent Fiber (USDA, Agricultural HandbookNo. 379). They are all gravimetric procedures and rely on filtration toisolate the fiber fraction. New fiber methods (Crude Fiber Analysis,AOCS method Ba 6a-05 and the ANKOM patent U.S. 5,370,007) have beendeveloped that use filter bags to improve the filtration step and enablebatch processing. AOAC refers to Association of Official AnalyticalChemists and AOCS refers to American Oil Chemists Society. In humannutrition Total Dietary Fiber (TDF) is the term used to classify fibercomponents that have certain nutritional and digestive tract benefits.Considerably different from the animal fiber methods, the TDF methodshave an additional requirement to precipitate the water soluble fiberfraction using alcohol. Thus fiber components that are water soluble inthe early enzymatic stages of the method are later precipitated withalcohol and recovered as a part of the fiber fraction.

AOAC Official Method 991.43 (one of the approved TDF methods) firstperforms an enzymatic digestion of the starch and protein in the sampleby treating it in a buffer solution with alpha amylase, then withprotease, and finally, after the appropriate pH adjustment, withamyloglucosidase (AMG). TDF consists of two components; an insolubledietary fiber (IDF) fraction and a soluble dietary fiber (SDF) fraction.TDF can be determined either by filtering the IDF and SDF fractionstogether in one filter, or by filtering the IDF and SDF fractionsseparately and then adding the two values together. In order to analyzethe IDF and SDF fractions separately, the IDF fraction is filtered atthe end of the enzyme digestion phase. Four volumes of ethanol are addedto the filtrate to precipitate the SDF fraction. The subsequentprecipitant is then separated by filtration. These filtrations arecommonly time consuming and difficult. The process requires a frittedglass crucible with a layer of diatomaceous earth and a vacuum system todraw the liquid through the filter. In many samples the IDF and the SDFtend to coat the filter and inhibit the liquid passage, requiringextended periods of filtration. To facilitate filtration, the surface ofthe diatomaceous earth filter pad often requires scraping.

Transferring the entire sample quantitatively at two different timesduring the analysis (once to the IDF filter and once to the SDF filter)is critical. A fine precipitation of the SDF fraction coats the beakerwalls and requires physical removal. The technician must take great careto scrape and rinse the beaker walls in order to transfer all of thefiber into the filtering crucible. Both the IDF and the SDF fractionsare recovered in the filtering crucible. The quantities are determinedgravimetrically by drying the crucible, weighing and subtracting theweight of the crucible (along with the diatomaceous earth filter pad),and correcting the sample for ash and protein. Those skilled in the artwill understand that correcting the sample for ash involves burning aduplicate sample and measuring the remaining ash. Correcting for proteininvolves analyzing a duplicate sample using the Kjeldahl or Dumas methodto measure the amount of protein in the sample. The IDF and SDF weightvalues are adjusted based on the ash and protein values. Total DietaryFiber can be calculated by adding the IDF and SDF values after they havebeen corrected for ash and protein.

As can be seen from the above description, the analysis of dietary fiberis a long and arduous procedure with problematic transfer and filtrationsteps. Accurate control of conditions and careful quantitative transfersare required by the technician to produce accurate and precise results.Every particle in the digestion flask must be transferred to thefiltering crucible and the diatomaceous earth filter pad must remainintact during the transfer in order to effectively capture the fineparticulate. Filtration for many samples is slow even with strong vacuumassistance. Some samples may require a scraping of the filter pad torenew some of the filter surface in order to complete the filtrationprocess. The scraping process must be accomplished without exposing thecourse filtering frit of the crucible. Many of the precision problemsassociated with this method are related to the difficulty of thetransfer and filter processes.

SUMMARY OF THE INVENTION

The application of this invention can be applied to multiple approvedTDF methods (e.g., AOAC 991.43 and 985.29). For purposes ofillustration, the present invention focuses on AOAC 991.43. The presentinvention is based on the discovery that a new procedure utilizing twofiltering containers may be employed in place of the standard in-vitroprocess described above. The time consuming, equipment intensivefiltering method of the prior art has been replaced in the presentinvention by two consumable containers that take the form of flexiblefilter bags. The first bag is the IDF bag which consists of a non-porousfilm with a porous filter in the lower part of the bag. Because a singlebag can be divided into reaction and filtration sections using atemporary seal or other temporary separation mechanism, the transferproblem of the prior art is solved by eliminating the step where atechnician manually transfers the mixture from a beaker to the filteringdevice. (It should be noted that in various embodiments a seal could bemade of a pinch mechanism, a clamp, a Smart Zip™ seal, a breakablemembrane, or any other mechanism that allows one section of thecontainer to be temporarily separated from another section.) Bytemporarily sealing just above the filter, the upper section is isolatedduring the enzymatic incubations. To assist the incubations, the uppersection is agitated and heated from the outside of the bag. Uponcompletion of the incubations the seal is released allowing the liquidto pass through the filter directly into the SDF bag. Filtration can beassisted by pressurizing the IDF bag.

The second bag is the SDF bag which also consists of a non-porous filmwith a porous filter in the lower part of the bag. By temporarilysealing above the filter, the SDF bag has an upper section that acts asa precipitation compartment, and a lower section that acts as a filtercompartment. The SDF bag's upper and lower sections are temporarilyisolated from each other until the precipitation process is complete.Because a single bag can be divided into precipitation and filtrationsections using a temporary seal or other temporary separation mechanism,the transfer problem of the prior art is solved by eliminating the stepwhere a technician manually transfers the mixture from a beaker to thefiltering device.

More specifically, the method involves an enzymatic digestion of asample within the IDF bag in a temperature and pH controlledenvironment. After the sample is digested the pinch mechanism isreleased allowing the mixture to flow into the filter section of thebag. Pressure is applied and the IDF fraction is captured in the filterwhile the filtrate containing the soluble SDF fraction flows directlyinto the upper non-porous section of the SDF bag that is preferablyprefilled with hot ethanol. Upon contact with the ethanol solution, thesoluble fiber is precipitated and is held for 60 minutes to complete theprecipitation. After the precipitation of the fiber the pinch mechanismis released allowing the mixture to flow into the filter section of thebag. Pressure is applied and the precipitate (SDF fraction) is capturedin the filter section of the bag. Both of the filter bags are rinsed anddried to ensure that only the respective fiber remains in the bags.Duplicate samples are used to determine protein and ash content. Thefinal IDF and SDF values are calculated using the weights corrected forash and protein content. When calculating the IDF and SDF fractionsseparately, total dietary fiber is determined by adding the correctedIDF and SDF fractions together. Total dietary fiber can also bedetermined without first determining the IDF and SDF fractions. In thiscase, the IDF bag is used only for the enzymatic digestions. After thedigestions are complete, the pinch mechanism is released and the entiremixture flows into the SDF bag without filtering the IDF fraction. Afterthe SDF precipitation phase, the mixture is filtered and rinsed. The SDFbag is then dried and weighed. Duplicate samples are used to determineprotein and ash content. In this case, the final TDF value is the weightof the TDF residue corrected for protein and ash content.

Both the IDF and SDF bags improve the analytical process by eliminatingthe need to transfer to the filtering crucible. Filter performance isalso improved by incorporating superior filter media and a largerfiltering surface area in the bags.

The primary advantages of the present invention are: (1) it does notrequire a technician to transfer the sample from a beaker to the filter;and (2) improved filtering capability eliminates the need for technicianintervention in the filtering process. Both of these advantages haveenabled cost saving automation of this method along with the potentialfor improved precision.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side sectional view illustrating an apparatus suitable foruse in carrying out the present invention.

FIGS. 2A-2E of the drawings schematically illustrate how the apparatusof FIG. 1 is used in each step of the process of the present invention.

FIGS. 3A-3B of the drawings illustrate another apparatus suitable foruse in carrying out the present invention consisting of a singlereaction/filtration container with multiple pinch points used toseparate the container into sections.

FIG. 4 of the drawings schematically illustrates how the apparatus ofFIG. 3 is used to automatically execute AOAC Method 991.43.

FIGS. 5A-5F of the drawings schematically illustrate how the apparatusof FIG. 3 is divided into a three section container and used in eachstep of the AOAC Method 991.43.

FIGS. 6A-6F of the drawings schematically illustrate how the apparatusof FIG. 3 is configured as a single section container and used in eachstep of the Crude Fiber method (AOCS method Ba 6a-05).

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows an apparatus that can be used to perform the process ofdigestion, precipitation, and filtering of a sample for the purpose ofdetermining IDF, SDF, and TDF as specified in AOAC Method 991.43. Theapparatus includes a removable IDF bag 1 consisting of an upper section2 made of a polymer film and a lower section 3 made of a porous filtermedia. The IDF bag is placed into the digestion vessel 4 with the top ofthe bag remaining open and folded over the vessel lip. A lid 10 closesonto the vessel lip creating a seal between the top of the bag and thevessel. Buffer and enzymes are introduced into the bag through port 8.Port 9 functions as a vent or introduces pressure to assist filtering. Astirring paddle 11 is used to mix the sample and the enzymes. A bandheater 12 and cooling coils 13 are used to maintain and control thetemperature of the vessel at 60° C. and 95° C. Pinch mechanism 15temporarily closes off a flexible tube 14 separating the liquid invessel 4 from the SDF bag 5. The SDF bag 5 consists of an upper section6 made of a polymer film and a lower section 7 made of a porous filtermedia. The SDF bag 5 is temporarily suspended from a manifold 19.Pressure is applied through port 18 into the SDF bag 5 to assistfiltering. Port 18 also acts as a vent port when ethanol is introducedthrough port 17. Pinch mechanism 16 temporarily separates the uppersection 6 from the lower section 7.

FIGS. 2A-2E of the drawings schematically illustrate how the apparatusof FIG. 1 is used in each step of the process of the present invention.In FIG. 2A an IDF bag 1 is placed into the digestion vessel 4 with thetop of the bag remaining open and folded over the top of the vessel.Pinch mechanism 15 is closed and a sample 20 is pre-weighed and insertedinto the IDF bag along with a buffer solution 21. SDF bag 5 is attachedto the manifold 19 and pinch mechanism 16 is closed, sealing the uppersection 6 from the lower section 7. The “x” objects in FIG. 2B throughFIG. 2D schematically illustrate the insoluble fiber, while the roundobjects in FIG. 2D and FIG. 2E illustrate the soluble fiber.

FIG. 2B illustrates that lid 10 is closed to seal the IDF bag. Thesample is dispersed in the buffer solution by mixing with paddle 11 andallowed to move freely throughout the entire bag. Amylase is added tothe vessel through port 8 and the mixture temperature is maintained at95° C. for 30 minutes. The temperature is then lowered to 60° C. Afteradding protease through port 8, the mixture is incubated for 30 minutes.By adding HCl through port 8, the pH is then lowered to a range between4.0-4.7. Amyloglucosidase is then added through port 8 for a finalincubation of 30 minutes. At the end of these enzyme digestions allcomponents are in solution except the insoluble fiber residue.

FIG. 2C illustrates the transfer of liquid from the IDF bag 1 to theupper section 6 of SDF bag 5. This is performed by opening pinchmechanism 15 and applying pressure through port 9 to enhance thesolution transfer through filter section 3. The non-fiber and solublefiber components that are in solution pass through filter section 3while only the insoluble fiber is retained. Through port 8 the retainedfiber is rinsed with 70° C. H₂O, 78% ethanol, and 95% ethanol. The uppersection 2 of the IDF bag provides a smooth wall to contain fiber solidswithout entrapping them. Because the fiber tends to slide off the filmwalls and collect in the lower filter section 3, rinsing can be doneefficiently. The upper section 6 of SDF bag 5 retains the solutionbecause it is constructed of a non-porous polymer film.

FIG. 2D illustrates the addition of 95% ethanol to SDF bag 5 throughport 17 with enough velocity to mix the solution. The addition of thefluids causes the bag to change shape. Over a period of 60 minutes thefiber fraction is allowed to precipitate and collect on the walls andbottom of the bag. As illustrated in FIG. 2E, after the precipitation iscomplete the pinch mechanism 16 is opened and the mixture is allowed toflow into the lower filter section 7 of SDF bag 5. The precipitatedfiber fraction is retained on both the walls of the upper section 6 andin the lower filter section 7 while the liquid filtrate flows throughthe filter and out of the bag. The walls and filter are rinsed with 78%ethanol, and 95% ethanol through port 17 to remove extraneous solublesfrom the fiber fraction. Because the fiber fraction is very fine, inmany cases it tends to adhere to the walls of SDF bag 5. However, sincethe entire SDF bag 5 is processed and weighed in later steps, removingthe fiber from the walls is not necessary. The IDF and SDF bags (withresidue) are removed, dried, and weighed. Duplicate IDF and SDF bags areanalyzed for ash and protein. The final IDF and SDF values arecalculated by subtracting the ash and protein weights from the weightsof the dried IDF and SDF residue in the bags.

FIG. 3 shows two different views of a removable flexible container madeof a polymer film with a porous filter media 26 in the bottom part ofthe container. In this figure, the container is shown to be pinched inthree different locations (22, 24, and 27) to create distinct sections(23 and 25). Although this drawing shows two distinct sections, it willbe understood by one skilled in the art that the container described inthis invention can consist of one, two, three, or more sections.

The containers used in the present invention can be produced using anytechnology that can create containers made of a non-porous polymer filmwith an attached porous filter media that provides for fine particleretention while allowing for rapid liquid passage. Depending on theapplication, the porous filter media can either be attached to theinside of the polymer film (allowing containment of the filteringprocess) or comprise the bottom part of the container as its outsidewall such that the top of the container is a polymer film and the bottomof the container is a filter material (allowing all filtrate to freelypass out of the container). To perform their function during theanalysis, the containers must also have characteristics that include: 1)the ability to be temporarily sealed to form one or more sections, and2) the ability to resist chemical breakdown under the temperature andpressure conditions of the analysis while also having the ability to beconsumed during the processes required to determine the ash and proteincontent of the sample. Suitable materials for the film part of the bagsinclude polypropylene, polyethylene, and polyester. Suitable materialsfor the filter sections of the bags include fiberglass, Teflon™,polypropylene, and polyester. In one embodiment polypropylene was usedfor the film portion and a melt blown polybutylene terephthalate wasattached to the inside of the polypropylene film and used as the filtermaterial. The typical bag will weigh up to about 100 g and be able tohold up to about 1000 cc of material.

Although this invention is primarily used in the determination of IDF,SDF, and TDF in foodstuffs, it should be understood that the presentinvention can also be used to determine IDF, SDF, and TDF in feedstuffs.Additionally, this invention can be used in any application where asolid is formed from a liquid and requires quantitative separation suchas various forms of precipitation, crystal formation, colloidalsolutions, and flocculation. Examples include purification of protein bysalting out, crystal formation of sugars due to temperature orconcentration changes, and bacterial flocculation due to the addition ofa flocculation agent.

EXAMPLES

FIG. 4 shows a schematic of an automated TDF system 30 where manifold 31is connected by appropriate tubing to the various liquid reservoirs(labeled individually) which supply the IDF and SDF bags with thenecessary chemicals and water through tubing 32 which is interconnectedto a heater 33 and pump 34. In operation, this system automaticallyexecutes AOAC Method 991.43 by using conventional electro-mechanicalcircuitry and hardware (not all shown), which is well known to those inthe art, to: 1) close the pinch mechanisms (to create the appropriatecontainer sections), 2) add the necessary chemicals (by moving them fromreservoirs through a plumbing system that includes a heater 33 and pump34), 3) heat and mix the sample with the chemicals (using a heater/mixer35 that is external to the container), and 4) open the pinch mechanismsto filter the solids and transfer the liquid between the sections of thecontainers. In the drawing, this automated system shows one IDF bag andone SDF bag. However, this system can be configured to process more thanone IDF bag and more than one SDF bag at once to allow for theconcurrent analysis of multiple samples (as shown on the drawing by theshort lines 36 extending out of the Heater). To execute AOAC 991.43using this automated instrument, weigh 1±0.005 g of a foodstuff samplein a pre-dried and tared IDF bag made of a polypropylene film with ameltblown polybutylene terephthalate filter capable of filteringparticles as small as 3-5 microns. Insert the IDF bag in the top part ofthe instrument and the SDF bag in the lower part of the instrument.Separate the digestion section 38 from the IDF filter section 40 byclosing pinch mechanism 39. Separate the precipitation section 42 fromthe SDF filter section 44 by closing pinch mechanism 43. Fill theinstrument reservoirs with MES-TRIS buffer, alpha amylase, protease,HCl, AMG, H₂O, 78% ethanol, and 95% ethanol. The automated AOAC 991.43method is initiated by turning the instrument on using the on/off powerswitch (not shown) and then pressing “Start” on the control paneldisplay (not shown). The instrument is designed to automatically addsolutions, maintain temperatures, mix the sample, and transfer solutionfrom the IDF bags to the SDF bags. The instrument performs the enzymedigestions inside the IDF bag section 38 maintaining proper temperatureand pH for optimum activity. Before the digestion phase is complete, hotethanol is introduced into the SDF bag precipitation section 42. At theend of the digestion phase pinch mechanism 39 separating the digestionsection 38 from the filter section 40 is opened and the mixture isfiltered with some pressure assistance (shown as N₂ in the drawing). Thefiber residue is retained in the IDF filter bag section 40 and thefiltrate flows into the SDF precipitation section 42. (It should benoted that precipitation is initiated as soon as the filtrate isintroduced into the SDF precipitation section 42 that is prefilled withethanol even though the IDF process is not fully complete.) IDF isrinsed with 70° C. H₂O, 78% ethanol, and 95% ethanol. When the IDFprocess completes, pinch mechanism 41 closes. After a one hourprecipitation period pinch mechanism 43 is opened and the mixture isallowed to flow into the SDF filter section 44. The filtrate passesthrough the filter with a pressure assist (shown as N₂ in the drawing)and the SDF is retained in the filter. The precipitated fiber is rinsedwith 78% ethanol and 95% ethanol. The IDF and SDF bags are removed fromthe instrument, rinsed with acetone, dried in an oven, and then weighed.Duplicate samples are used to determine protein and ash content. Thefinal IDF and SDF values are calculated using the weights corrected forash and protein content. In this example TDF can be calculated by addingthe corrected IDF and SDF values.

In addition to its use in the IDF and SDF procedures previouslydiscussed, the conceptual principle of this invention can bedemonstrated in other analytical techniques illustrated by the followingexamples.

It will be understood by one skilled in the art that the reaction/filtration container described in this invention can be divided into oneor more sections. In an application of the invention, the AOAC 991.43TDF analysis can be performed in one flexible filter bag assembly (FIG.5) divided into three sections isolated by pinch mechanisms. In thisexample the upper section 45 is used as the digestion/reactioncompartment, the middle section 46 is used as the precipitationcompartment, and the lower section 47 is used as the filter compartment.In FIG. 5A, the bottom pinch mechanism 48 is closed and hot ethanol isadded to the container. This is done to eliminate the need for assistedmixing during the precipitation phase. In FIG. 5B, the middle pinchmechanism 49 is closed and the appropriate chemicals are added tosection 45. In FIG. 5C, the top pinch mechanism 50 is closed and section45 is heated and agitated by an external source for the required time toperform the three enzymatic digestions. When the enzymatic digestionsare complete the middle pinch mechanism 49 is opened and the digestionmixture drops into the hot ethanol (FIG. 5D). After the soluble fiber isfully precipitated (FIG. 5E) the lower pinch mechanism 48 opens (FIG.5F). The filter retains the IDF and SDF while the liquid passes through.The filtration can be assisted by adding pressure through the top pinchmechanism 50 (shown as N₂ on the drawing). After the rinsing proceduresare completed the entire bag is dried and weighed. The TDF value can becalculated after determining suitable corrections for ash and protein.

In another application, the reaction/filtration bag 53 can be configuredas a one section container with a filter material inside for theanalysis of Crude Fiber (AOAC method Ba 6a-05). This is accomplished inFIG. 6 by closing pinch mechanism 51 below the filter material 54. Thisallows for sequential treatments of a solid sample. For Crude Fiberanalysis, the sample can be placed in the bag along with the acidsolution and the bag sealed at 52. The mixture can be heated andagitated for the appropriate amount of time. When the time has elapsed,pinch mechanism 51 is opened (FIG. 6C) allowing the liquid to flow out.Pinch mechanism 51 is then re-closed below the filter (FIG. 6D). Thebase solution is added to the bag, pinch mechanism 52 is closed, and themixture is again agitated and heated for the appropriate amount of time(FIG. 6E). When the time has elapsed, pressure assist is added (shown asN₂ on the drawing) and pinch mechanism 51 below the filter is openedallowing the liquid to flow out (FIG. 6F). After rinsing, the bag isdried and Crude Fiber is determined gravimetrically.

In another application the reaction/filtration bag can be used todetermine the degree of fermentation of beer (AOAC method 950.06). Beer(250 ml) is added to the upper section of the bag with 1 g activecompressed brewer's yeast and fermented for 24-48 hours at 15-25° C. Theupper section is left slightly open on the top to allow pressure to bereleased. A pinch seal that separates the reaction in the upper sectionfrom the lower filter section is opened and the solids are retainedwhile the liquid is collected in a separate flask. The difference inspecific gravity is determined before and after fermentation of thebeer. Fermentable sugars are equal to the difference multiplied by 0.82.

In another application the reaction/filtration bag can be used todetermine water soluble solids in roasted coffee in an adaptation ofAOAC method 973.21. Roasted coffee sample (10 g) is placed in the uppersection with 200 ml of water. The bag is sealed above and below theupper section and heated from the outside to 100° C. Pressure is allowedto climb to 2-4 psi to suppress boiling. After 5 minutes at temperaturethe seal at the bottom of the upper section is opened. The solids arecollected in the lower filter section. The water soluble coffee fractionis determined by dry matter disappearance. The bag and its contents aredried at 105° C. and weighed. The amount of soluble fraction extractedis determined by the loss of weight of the sample.

In another application, the reaction/filtration bag can be used in thepreparatory phase of a spectrophotometric procedure for the analysis ofcarbodox in feeds (AOAC method 977.35). A 5 g sample is placed in theupper section and 10 ml of water is added to wet the sample. After 5minutes, a 3:1 mixture of chloroform and methanol (140 ml) is added tothe upper section. The section is heated to its boiling temperature for1 hour. The contents are allowed to cool to room temperature and thenallowed to pass through the filter. The filtrate is further processedand the absorbance is measured at 520 nm to determine the quantity ofcarbodox.

In another application, the reaction/filtration bag can be used in thepreparatory phase of a liquid chromatographic procedure for the analysisof diquat and paraquat residues in potatoes in an adaptation of AOACmethod 992.17. Add 5 g of macerated potato to the upper section of thebag with 5 ml of 2N HCl. The bag is sealed above and below the uppersection with a pinch seal, heated to 100° C., and agitated for one hour.The seal at the bottom of the upper section is opened and solids arecollected in the lower section filter. The solids captured in the filterare then rinsed with 6 ml of H₂O and the filtration is enhanced bypressurizing the upper section. The bottom portion of the imperviousfilm that extends past the filter is then closed mechanically andanother 5 ml of 2N HCl is added, heated, and agitated for 30 minutes.The seal beneath the filter is released, the solution is filtered andthe filtrates are further processed to prepare for chromatography. Theanalysis is completed on a poly (styrene-divinylbenzene) column with anultraviolet detector (254 nm & 313 nm filters).

While the present invention has been particularly shown and describedwith reference to the preferred mode as illustrated in the drawings, itwill be understood by one skilled in the art that various changes indetail may be effected therein without departing from the spirit andscope of the invention as defined by the claims.

We claim:
 1. A flexible bag used for analyzing a sample having aninsoluble fraction and a soluble fraction comprising: a reaction sectioncomprising a non-porous polymer film for combining the sample with aliquid; and a filtration section comprising a porous filter forfiltering the sample after combining in the reaction section, whereinthe porous filter is attached to the inside of the non-porous polymerfilm of the reaction section, wherein a portion of the non-porouspolymer film of the reaction section surrounds and contains a portion ofthe porous filter.
 2. The flexible bag of claim 1, wherein thenon-porous polymer film of the reaction section is integrally formedwith the porous filter of the filtration section.
 3. The flexible bag ofclaim 1, wherein the reaction section has a smooth wall to contain theinsoluble fraction without entrapping it.
 4. The flexible bag of claim1, wherein the non-porous polymer film is polypropylene, polyethylene,or polyester.
 5. The flexible bag of claim 1, wherein porous filter ismade of fiberglass, Teflon, polypropylene, polyester, or melt blownpolybutylene terephthalate.
 6. The flexible bag of claim 1, wherein theflexible bag weighs 100 g or less.
 7. The flexible bag of claim 1,wherein the flexible bag can hold 1,000 cc of material or less.